Toumatia et al. 2015.PDF

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Effectiveness of a new actinomycin D-producing strain for biocontrol

time favorable for active compound production. Three
milliliters of seed culture was prepared with the same
medium, incubated at 30 °C for 2 days and used to
inoculate 500-ml Erlenmeyer flasks containing 100 ml of
medium. The cultures were incubated on a rotary shaker
(250 rpm) at 30 °C. The antimicrobial activity of the
culture broth was monitored by the conventional agar
diffusion assay (well technique) against B. cinerea and
F. oxysporum f. sp. lini.
Extraction and purification of the bioactive compound
Preparative chromatography with silica gel plates (Merck
Art. 5735, Kiesselgel 60HF 254-366; 20 cm 20 cm) was
employed for the partial purification of antimicrobial
products. TLC plates were developed in the ethyl acetate–
methanol solvent system, 100:15 v/v. The developed TLC
plates were air dried overnight to remove all traces of the
solvents. The compounds separated were visualized with
the naked eye, under UV at 254 nm (absorbance) and at
365 nm (fluorescence). The bioactive spot was detected by
bioautography [24] toward the two previously cited target
fungi. The retention factor (Rf) of the bioactive spot
was measured. The final purification of the most active
compound (YA) was performed by HPLC on reverse phase
XBridge C18 (5 mm) column (200 mm 10 mm; Waters,
Milford, MA) with a linear gradient of acetonitrile–H2O
(50–100% for 40 min), a flow rate of 1 ml min 1 and UV
detection at 220 nm. The final purification was achieved
after the second re-injection in the HPLC system.
Determination of the bioactive compound YA structure
The structure of the compound YA was mainly elucidated
with the aid of spectroscopic investigations. The UV
spectrum was given with a Shimadzu UV 1605 spectrophotometer. The mass spectrum was recorded on an LCQ
ion-trap mass spectrometer (Finnigan MAT, San Jose, CA)
with nanospray ion electro-spray ionization (ESI) source
(positive and negative ion mode). 1H and 13C NMR
spectroscopy were used for the characterization of the
active molecules. The NMR sample was prepared by
dissolving 3 mg of purified compound in 600 ml of
CD3OD. All spectra were recorded on a Bruker Avance 500
spectrometer equipped with a 5-mm triple-resonance
inverse Z-gradient probe (TBI 1H, 31P, BB). All the
chemical shifts for 1H and 13C were relative to TMS using
H (residual) or 13C chemical shifts of the solvent as a
secondary standard. The temperature was set at 298 K.
All the 1H and 13C signals were assigned on the basis of
chemical shifts, spin–spin coupling constants, splitting
patterns, and signal intensities, and by using 1H-1H
COSY45, 1H-13C HSQC, and 1H-13C HMBC experiments.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim


Gradient-enhanced 1H COSY45 was realized included
36 scans per increment. 1H-13C correlation spectra using
a gradient-enhanced HSQC sequence (delay was optimized for 1JCH of 145 Hz) were obtained with 200 scans
per increment. A gradient-enhanced HMBC experiment
was performed allowing 62.5 ms for long-range coupling
evolution (340 scans were accumulated). Typically, 2048
t2 data points were collected for 256 t1 increments.
Biocontrol properties of the strain IA1
The strain IA1 and its highly produced bioactive
compound named YA (yellow antibiotic) were assessed
in biocontrol trials. Biocontrol abilities were evaluated
through two different plant–pathogen disease systems:
chocolate spot of field bean and Fusarium wilt of flax
caused by B. cinerea and F. oxysporum f. sp. lini, respectively.
Seed material
Seeds of field bean (Vicia faba L., variety Giza 429) and flax
(Linum usitatissimum L., variety Hera) were supplied by the
Technical Institute of Field Crops, Algiers, Algeria. Prior
to use, all seeds were surface-sterilized (5% w/v NaClO;
0.2% w/v Tween 20) for 3 min for flax seeds or 15 min
for field bean, and then rinsed five times with sterile
distilled water.
Fungi and strain IA1 inocula preparation
Pathogenic fungi strains, isolated from diseased plant
culture of flax and field bean, were supplied by the
Department of Botany, High School of Agriculture,
Algiers, Algeria. Prior to use, fungi isolates were
subcultured on potato dextrose agar (PDA) plates and
incubated at 25 °C for 7 days. Strain IA1 was grown on ISP2 medium plates and incubated at 30 °C for 10 days.
Suspensions of both fungal conidia and actinomycete
spores were obtained by scraping from the culture
surface with a glass slide, homogenized in sterile distilled
water (0.2% w/v Tween 20) and filtered through a double
layer of sterile gauze [16]. The concentrations were
adjusted by hemocytometer chamber counting method.
Plant growing conditions
Seedlings were placed in a phytotronic growth chamber
with 80% relative humidity, a temperature of 22 3 °C
and 14 h of light (8000 Lux) period conditions.
Biocontrol assay of chocolate spot disease
Field bean seeds were first pre-germinated in Petri dishes
containing sterile wet paper for 3 days in darkness at
15 °C, than sown in pots (five seeds per pot, 1 cm depth)
filled (100 g per pot) with sterile rhizospheric soil
(autoclaved at 120 °C for 20 min, three times, once

J. Basic Microbiol. 2014, 54, 1–8